The interaction of molecular species with cellular targets is critically important in understanding cellular physiology and developing therapeutic interventions, such as new synthetic drugs and biopharmaceuticals. Methods are needed for accurately and efficiently determining target engagement, particularly within living cells where these interactions mediate their phenotypic responses. The ability to affectively interrogate target engagement has broad implications to the discovery process, ranging from high-throughput screening, optimization of screening hit into drug leads, and the discovery and characterization of therapeutically relevant cellular targets.
Phenotypic-based screening with a small molecule library plays an important role in the drug discovery field. Using such screening approaches, compound libraries, without prior knowledge of their underlying cellular targets, are screened for their ability to elicit a phenotypic response, e.g., mitigate disease symptoms. While this approach can be used to identify bioactive agents, e.g., small molecules, that are able to modulate cellular physiology, determining the biological relevant targets of these small molecule hits is a major technical challenge. In addition, small molecules promoting some desirable phenotypic responses may pose in vivo liabilities due to off-target interactions. In order to predict drug selectivity and minimize potential side effects, it is important to also identify off-target interactions (e.g., lower affinity). Most methods used today for identifying the targets of bioactive agents rely on the enrichment of these targets from complex cellular lysate using “bi-functionalized” compounds that contain a selective moiety (e.g., the bioactive agent or related compound) and a sorting moiety (e.g., affinity tag or solid support). As the enrichment is based on the binding properties of the compounds, where the inherent affinity of these compounds for their target is insufficient, compound analogs are designed to covalently bind to the target (e.g., photo-crosslink). By either approach, the efficacy and specificity of target isolation is essential, and the failure rate of these methods is high. Failure of these approaches can be due to insufficient capture of the target or high background due to non-specific capture. Contributing factors to this failures include: compounds binding multiple targets with low to moderate affinity with these relative weak interactions difficult to detect; lack of robust, straightforward, unbiased technologies to characterize the detected interactions; inability to perform target isolation within the native cellular environment upon which interactions may depend; limited information provided about the binding potency of targets in the cell; and high background of false positive interactions due to non-specific binding to the solid support or functionalized small molecule.